In a first phase of efforts, we discovered that the Tabby mouse, which has many of the features observed in human EDA, is specifically mutated in the corresponding mouse gene. We demonstrated that the Wnt pathway directly regulates EDA transcription. In published work, we further found that provision of DNA encoding a variant of ectodysplasin (Eda-A1, the longest isoform) in embryonic Tabby mice restores hair follicles and sweat glands. By generating Tet-regulated conditional transgenic mice, we have dissected spatiotemporal actions of Eda-A1 during hair follicle development. We also have characterized eye phenotypes of Tabby mice including blindness and inflammation susceptibility, and they are also reversed by supplementation with the same Eda-A1 isoform. This study provided the first animal model for ocular surface disease. We identified numerous downstream target genes of Eda. More recently, we have further focused on the function of Eda and Eda target genes in mutant mouse models. One puzzle in developmental biology is that certain signaling pathways are often involved in development e.g., Wnt, Shh, and Bmp in skin appendage formation, but how can the same pathways lead to different final tissues? To resolve this puzzle, we are now focusing on two skin exocrine glands, sweat glands and Meibomian glands, again aided by Tabby mice as a model. We are also focusing on how the final secretion of sweat or Meibomian oil is regulated. Analysis of cascade regulation of sweat gland development by major morphogenetic signaling pathways including Eda. Wnt/beta-catenin is required for Eda activation and the initiation of sweat gland formation. In Tabby mice, where Eda signaling is abolished, we found that sweat gland germs then started to form but aborted at pre-germ stage. Eda action is also required for sweat duct formation, followed by Shh action.. Additional layers of regulatory mechanism of sweat gland formation: Involvement of small non-coding RNAs in sweat gland development. miRNAs are known to act negatively for mRNA stabilities and their translation. We are currently analyzing miRNA function in sweat gland development. We generated skin specific Dicer knockout mice, and found that sweat glands were not formed. We further found that miRNAs are required for sweat gland development downstream of Wnt and Eda. Studies have been extended to understand the secretory mechanism of sweat glands. Among genes expressed in sweat glands at different stages, FoxA1 was strikingly affected gene in Tabby during late developmental and adult stages. Skin-specific FoxA1 knockout mice showed striking anhidrosis, with abundant accumulation of glycoproteins in the lumens and ducts of otherwise complete sweat glands; and we further showed that FoxA1 functions in sweat glands by promoting transcription of an anion channel protein, Best2. Best2 knockout mice also showed severe hypohidrosis/anhidrosis, revealing a FoxA1-Best2 cascade as a fundamental genetic pathway in sweat glands, regulating sweat secretion. Because Best2 is a calcium activated bicarbonate channel, we inferred two alternative cascades for sweating: calcium K/Cl (FoxA1) additional monovalent ion transporter cascade and calcium (FoxA1) Best2 K/Cl ion transporter, with the latter likely playing the major role. We further found that Foxc1, another Fox family transcription factor, is also highly expressed in mouse sweat glands. To analyze Foxc1 function in sweat glands, we imported Foxc1-loxP mice recently from Dr. Kumes lab in Northwestern Univ., and generated skin specific Foxc1 knockout mice. Foxc1 cKO mice showed severe hypohidrosis, however, sweat glands were fully formed. In more detailed analysis, we found that sweat ducts were blocked by hyperkeratotic or parakeratotic plugs, which was strikingly similar to human miliaria sweat retention disorder. The phenotypes suggested that Foxc1 is not required for sweat gland development, but affects sweat secretion. By expression profiling, we found a set of keratinocyte terminal differentiation related genes were significantly upregulated in Foxc1 cKO sweat glands, and among them at least Sprr2a/b and Krt8 were ectopically expressed in sweat duct luminal cells in the cKO mice. By promoter analysis, we further found that Foxc1 negatively regulates Sprr2a expression in keratinocytes. Therefore, we could demonstrate that Foxc1 represses terminal differentiation related genes in sweat ducts in normal condition, and Foxc1 ablation causes ectopic expression of those differentiation related genes resulting in keratotic plug formation and hypohidrosis. We are also analyzing function of individual ion channels in sweat glands. K+ and Cl- channel opening is thought to be the first ionic event in sweat secretion. We found 6 K+ and Cl- channels in mouse sweat glands. We further found that simultaneous ablation of Kcnn4 and Kcnk5 in mice causes hypohidrosis. We are now carrying out single cell RNA-Seq analyses to define the number of sweat gland cell types and their gene expression complement. . Meibomian glands are specialized glands that lubricate the ocular surface. Tabby mice and EDA patients lack meibomian glands, and are thus susceptible for dry eye. We previously showed that Eda-ablated Tabby mice develop ocular surface disease, and EDA patients show extreme dry eye. We plan to examine dry eye etiology with meibomian glands as an entry point. In a first phase, we characterized their development. They are missing in Tabby mice, and Shh knockout mice, Dkk4 transgenic mice, and beta-catenin knockout mice all completely lack meibomian glands. We have characterized meibomian gland phenotypes in these mice more systematically by time-course histological and immunohistochemical analyses. Regarding the initial action of signaling pathways in Meibomian glands, it was known that Eda acts through NF-kB transcription factors, but we have now shown that 2 specific ones of the 5 NF-kB factors are activated by Eda, and that they act in conjunction with a chromatin remodeling complex to achieve specific activation of target genes. In additional work, we have shown that Wnt inhibitor Dkk4 specifically inhibits Meibomian gland formation in a transient fashion, alleviated when it is proteoloytically cleaved. We have also now begun to examine aging-related effects in mice and find that for sweat glands, there is a marked decline in sweating capacity, with a reduction in the number of sweat glands.